Quality and quantity of DNA was measured using the Genomic DNA Screen Tape Assay (Agilent Technologies, Santa Clara, CA) and Qubit. The concentration of genomic DNA (gDNA) larger than 200 bp was then calculated, and at least 200 ng of DNA greater than 200 bp was sheared in 50 µL of nuclease-free water with the Covaris E220 using the 96 microTUBE Plate (Covaris, Woburn, MA). The library was prepared using KAPA Hyper Prep Kit (Roche, Basel, Switzerland) and 100–500 ng of sheared DNA according to the manufacturer’s instructions. Individual tumor adapter-ligated libraries were enriched into the exome capture reaction, and for germline each adapter-ligated library was pooled before proceeding to capture using Agilent’s SureSelect Human All Exon V6 + custom probes capture library kit [28 (link)]. Samples that had successful libraries created were then sequenced on Illumina MiSeq technology for quality control to assess the ability of the libraries to be sequenced. Subsequently, each library was pooled and sequenced on Illumina’s NovaSeq 6000 (Illumina, San Diego, CA) using 300 cycle kit. Raw FASTQs were generated using the industry standard BCL2FASTQ v1.8.4
96 microtube plate
Manufactured by Covaris
Sourced in United States
The 96 microTUBE Plate is a laboratory equipment product designed for use with Covaris instrumentation. It features 96 individual sample tubes, each with a 500 μL capacity. The plate is constructed from a durable, high-quality material to provide a reliable and consistent platform for sample processing.
Automatically generated - may contain errors
Lab products found in correlation
Genomic dna screentape assay, by Agilent Technologies (2 mentions)
Novaseq 6000, by Illumina (2 mentions)
Miseq technology, by Illumina (1 mentions)
Kapa hyper prep kit, by Roche (1 mentions)
Lightcycler 480 instrument 2, by Roche (1 mentions)
Le220 focused ultrasonicator, by Covaris (1 mentions)
Sureselectxt low input, by Agilent Technologies (1 mentions)
3 protocols using 96 microtube plate
1
Exome Sequencing Workflow for Tumor and Germline Analysis
2
DNA Fragmentation and Library Prep
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Quality control of input genomic DNA samples was conducted by visual inspection for discoloration and/or presence of precipitants. Genomic DNA quantitation was performed using a fluorescence dye-based assay (PicoGreen dsDNA reagent) and measured by a microplate reader (Molecular Devices SpectraMax Gemini XS) before normalization to 20 ng/uL. Normalized gDNA samples were added into wells of a Covaris 96 microTUBE plate at 55 μL volume and sheared using the Covaris LE220 Focused-ultrasonicator with settings for targeting a peak size of 410 bp (PIP: 450 W, Duty Factor: 18%, Cycles per burst: 200, Time: 60s). Sequencing libraries were generated from 1,000 ng of fragmented DNA using the Illumina TruSeq DNA PCR-Free HT Library Preparation Kit with minor modifications for automation on a Hamilton STAR Liquid Handling System. Adapters for ligation used either TruSeq DNA CD Indexes or IDT for Illumina TruSeq DNA UD Indexes (96 Indexes, 96 Samples). Library size distribution and absence of free adapters and/or adapter dimers was assessed by automated capillary gel-electrophoresis (Advanced Analytical Fragment Analyzer). Library yield and concentration (in nM) was determined by qPCR quantitation using the KAPA qPCR Quantification Kit on a Roche Light Cycler 480 Instrument II.
3
Comprehensive Genomic DNA Sequencing Protocol
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The quality and quantity of isolated DNA was measured using the Genomic DNA Screen Tape Assay (Agilent Technologies, Santa Clara, CA, USA). A 200-nanogram measure of DNA was sheared in 50 µL of nuclease-free water with a Covaris E220 using a 96 microTUBE Plate (Covaris, Woburn, MA, USA), followed by SureSelect XT Low Input (Agilent) with unique dual adapters. Adapter-ligated libraries were enriched using a custom Agilent’s SureSelect v6 + UTR + OneSeq LowRes (Agilent, Santa Clara, CA, USA) probe set. These additional custom probes (105 Mb exome) provided (1) evenly spaced probes for more-informative copy-number analysis and (2) probes that tile across non-coding regions for improved ancestry calling. Each library was normalized, pooled, and sequenced on Illumina’s NovaSeq 6000 using the S4 300 cycle flow cell (Illumina, San Diego, CA, USA) v1.0 chemistry. All sequencing reads were converted to industry-standard FASTQ files using BCL2FASTQ v1.8.4.
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